Vol. 29, No. 3

JoURNAL OF VIROLOGY, Mar. 1979, p. 1014-1022 0022-538X/79/03-1014/09$02.00/0

Initiation of DNA Synthesis on the Isolated Strands of Bacteriophage fl Replicative-Form DNA MARVIN L. BAYNEt AND LAWRENCE B. DUMAS* Department of Biochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201 Received for publication 24 October 1978

Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage fl replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on fl viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of fl viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on fl complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the fl replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for fl DNA replication in vivo proposed by Horiuchi and Zinder. The study of the conversion of the singlestranded circular DNA molecules of filamentous bacteriophages fl, fd, and M13 to their doublestranded replicative forms has been a useful model for the investigation of the de novo initiation of DNA synthesis in Escherichia coli. Reconstitution ofthis replication process in vitro has led to the isolation and characterization of the E. coli proteins involved in filamentous phage parental replicative form (RF) synthesis (11, 14). These studies revealed that DNA synthesis on the infecting viral strand circular DNA molecule is initiated through the synthesis of a short RNA transcript that serves as an oligonucleotide primer (26). The E. coli DNA-dependent RNA polymerase catalyzes the synthesis of this primer molecule (11, 14). In addition to DNA-dependent RNA polymerase, the E. coli dnaB and dnaG proteins appear to be required for the replication of filamentous phage duplex RF DNA in vivo (5, 21, 23). Recent in vitro studies have shown the E. coli dnaG protein to be a rifampin-resistant RNA polymerase (2). It is able to catalyze the synthesis of short oligonucleotides that initiate DNA synthesis on templates such as the singlestranded circular DNA molecules from bacteriophages 4X174 and G4 (24, 33). A pre-initiation complex that includes the E. coli dnaB protein is required for the dnaG protein-catalyzed syn-

thesis of the oligonucleotide primer on 4X174 DNA but not G4 DNA (2, 20, 33, 34). Both in vivo and in vitro studies indicate that these initiation proteins are unable to substitute for the E. coli DNA-dependent RNA polymerase in the synthesis of the primer oligonucleotide on the viral strand circular DNA molecules from filamentous phage particles (3, 25, 34). This suggests that the viral strand from phage particles cannot be recognized by these site-specific proteins. We were interested in determining if the in vivo requirements for the E. coli dnaB and dnaG proteins during filamentous phage RF DNA synthesis could be explained by their direct participation in the synthesis of a primer oligonucleotide on either the viral or complementary of the RF DNA molecule. We therefore isolated viral and complementary strand circular DNA molecules from bacteriophage fl RF DNA. These molecules were then used as templates for DNA synthesis catalyzed by soluble protein extracts of E. coli. Using this assay system, we indirectly examined oligonucleotide primer synthesis on the individual strands of fl RF DNA.

t Present address: Mergenthaler Laboratory for Biology, The Johns Hopkins University, Baltimore, MD 21218. 1014

MATERIALS AND METHODS Bacteria and phage strain. E. coli H560 (F+ poUA, endoiP, thy-) was obtained from A. Kornberg. E. coli PC79 (F- his, str- maU4, xyt, mtl, tht, poLA, sup , duaD7 [now referred to as dnaC]) was obtained from P. Carl. E. coli K38 and phage fl were

VOL. 29, 1979

DNA SYNTHESIS ON STRANDS OF fl RF DNA

obtained from N. Zinder. Phage G4 was obtained from N. Godson. E. coli C and phage OX174am3 were from stocks in this laboratory. Preparation of soluble protein extract of E coli E. coli H560 was grown and harvested according to the methods of McHenry and Kornberg (17). The preparation of an extract from gently lysed cells (fraction I) and the fractionation of the extract with ammonium sulfate (fraction I) were performed as described by Schekman et aL (25). Preparation of phage DNA. Bacteriophages oX174, G4, and ft were purified from infected E. coli by differential centrfugation. Phage proteins were removed by phenol extraction. The DNA was further purified by zone sedimentation in either neutral pH or alkaline pH high-ionic-strength sucrose gradients. Phage fl viral and complementary strand circular DNA molecules were isolated from intracellular supercoiled fl RFI DNA molecules as previously described (1). Analysis of the final DNA preparations indicated that at least 95% of the DNA was in the single-stranded circular form and contaminated by less than 5% of the opposite strand. Enzymes. E. coli DNA polymerase I was isolated according to Dumas et al. (6) and was a gift from J. Bieker of this department. Endonuclease B8t 1503 was prepared according to Catterall and Welker (4) and was a gift from N. Welker of this department. Endonucleases Pst I and Hae II were purchased from Bethesda Research Laboratories. Standard initiation assay. DNA synthesis was measured at 30°C in a standard incubation mixture of 50 pl containing: 40 mM Tris-hydrochloride (pH 7.5); 5 mM MgCl2; 4 mM dithioerythreitol; 4 mM spermidine; 0.8 mM ATP; 50 IM each dATP, dCTP, and dGTP; 22 pM [3H]TTP or [nP]TTP; 100 uM each GTP, UTP, and CTP; 250 to 350 pmol of singlestranded DNA (expressed as nucleotide residues); and approximately 0.2 mg of soluble protein extract fraction I. In some cases rfampin was added at 8 ug/ml. At the indicated times, 10-pl aliquots were removed and added to 0.2 ml of 0.1 M sodium pyrophosphate containing 0.2 mg of calf thymus DNA. Three milliliters of 5% trichloroacetic acid was added. The precipitate was collected on glaws fiber filters (Whatman GF/A), washed three times with 5% trichloroacetic acid and once with methanol, and dried. Three milliliters of toluene-based scintillation fluid was added and the filters were analyzed in a liquid scintillation counter.

Sucrose gradient analysis. Procedures for the isolation and characterization of nucleic acids by isokinetic alkaline and neutral pH sucrose gradients were as previously described (1, 7). Equilibrium gradient analysis. Viral and complementary strand ft DNA molecules were separated by alkaline CsCl equilibrium gradients by a modification of the method of Ray (22). The DNA preparation was adjusted to 4 ml in a solution of 0.2 N NaOH, 0.1% Sarkosyl, and 5 mM EDTA. Solid CsCl was added to a density of 1.755 g/ml. The solution was then spun for 68 h at 32,000 rpm at 5C in a Beckman type 40 rotor. The contents of the tube were displaced at 0.2 ml/min, and 3-drop fractions were collected. The frac-

1015

tions were analyzed for radioactivity as previously described (7). Preparation of fragment primed fl complementary strand DNA. The viral strand of endonuclease Hae II fiagment C from fl RF DNA (32) was annealed to fl complementary strand circular DNA by using a modification of the method of Edgell et al. (8). Endonuclease Hae II fiagment C was prepared by digesting 70 pg of fl RFI DNA with 40 U of enzyme at

370C in a total volume of 200 id containing: 50 mM Tris-hydrochloride (pH 7.5); 0.4 mM dithioerythreitol; 5 mM MgSO4; and 16 pg of gelatin per ml. After 4 h

the reaction was terninated by the addition of EDTA to 20 mM. The digested RF DNA was layered onto two isokinetic neutral pH sucrose gradients and spun for 8 h at 55,000 rpm at 15°C in a Beckman SW56 rotor. The gradients were displaced at 0.5 ml/min, and the absorbance at 260 nm was continuously monitored. Under these conditions, fiagment C (320 base pairs) was easily resolved from firgment A (3,500 base pairs) and faginent B (2,600 base pairs) (32). Fractions conent C were pooled and dialyzed against taining f 50 mM Tris-hydrochloride (pH 8.1)-2 mM EDTA. Fragment C (approximately 250 ng) was diluted to 50 id and denatured in a boiling water bath for 3 min. Complementary stand cicular fl DNA (1.2 pg in 200 id of 50 mM Tris-hydrochloride [pH 7.5]-2 mM EDTA-0.1 M NaCl) was added, and the mixture was incubated for 30 min at 570C, then for 22 h at 250C. Between 50 and 60% of the circular complementary strand molecules were primed by this procedure, as determined by template-primer activity measurements at 150C using E. coli DNA polymerase I in the presence and absence of random calf thymus DNA initiator fragments. Localization of the gap in ft RFU DNA. The gap in the complementary product strand of in vitrosynthesized fl RFII molecules was mapped by a modification of the method of Tabak et al. (30). Phage fl RFII molecules were synthesized using the standard initiation assay described above. After 10 min the reactions were stopped by the addition of EDTA to 10 mM and the addition of Sarkosyl to 0.5%. The RFII molecules were then purified in high-ionic-strength isokinetic neutral pH sucrose gradients. The RFII molecules were denatured and spun in isokinetic alkaline sucrose gradients. The majority of the 3P-labeled product sedimenting as unit-length linear DNA was pooled, and excess viral strand circular DNA molecules were added. This mixture was dialyzed at 40C against a solution of 50 mM Tris-hydrochloride (pH 8.1), 1 M NaCl, and 5 mM EDTA. The RFII molecules formed during dialysis were separated from remaining single-stranded DNA by zone sedimentation at neutral pH. This purified 3P-labeled RFII DNA was digested with endonuclease Bst 1503 in a final volume of 160 pl containing. 50 mM Tris-hydrochloride (pH 7.5); 0.8 and 2 plg of unlabeled fl RFI DNA; 0.2 mM MgSO4; mM dithioerythreitoL After incubation at 556C for 1 h, 20 id was removed and analyzed by agarose gel electrophoresis in the presence of ethidium bromide to confirm that the digestion had gone to completion. The digested fl RFII DNA was next layered onto a

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BAYNE AND DUMAS

isokinetic alkaline pH sucrose gradient and spun for 6 h at 55,000 rpm at 150C in a Beckman SW56 rotor. The gradients were fractionated and analyzed as described above. Infectivity assay. The infectivity of fl DNA molecules was determined by the spheroplast method described by Sinsheimer (27). Repair synthesis at 156C using E. coli DNA polymerase I. Single-stranded DNA molecules were copied by E. coli DNA polymerase I using calf thymus DNA fragments as random oligonucleotide primers by a modification of the method of Dumas et al. (6). Reactions were carried out in a final volume of 60 Ad containing: 20 mM Tris-hydrochloride (pH 7.5); 50 LM each dATP, dCTP, and dGTP; 8 MuM [3H]TTP; 2 mM dithioerythreitol; 10 mM MgCl2; 250 to 350 pmol of DNA (expressed as nucleotide residues); 60 nmol of degraded calf thymus DNA; and E. coli DNA polymerase I. At the indicated times, 10-p1 aliquots were removed, and acid-insoluble nucleotides were determined as described above.

sized in 30 min in the presence of rifampin, indicating the requirement for E. coli DNA-dependent RNA polymerase in the initiation of DNA synthesis on both viral strand templates. The product of DNA synthesis under these conditions was duplex, relaxed fl RFII DNA as judged by zone sedimentation analysis at neutral pH (Fig. 2A). Zone sedimentation analysis at alkaline pH demonstrated that the newly synthesized DNA strand of the RFII molecule was unit-length linear DNA (Fig. 2B). The location of the gap in the newly synthesized complementary DNA strands was determined by a modification of the procedure of Tabak et al. (30). For this analysis, purified fl RFII DNA molecules resulting from DNA synthesis on fl viral strand templates isolated from phage particles, and on fl viral strand templates isolated from intracellular RF DNA, were digested with endonuclease Bst 1503. This endoRESULTS nuclease is an isoschizomer of endonuclease DNA synthesis on fl viral strand DNA BamHI (4) and cleaves fl RF DNA at a single isolated from intraceihular RF DNA. Viral and complementary strand circular DNA molecules isolated from intracellular fl RF DNA molecules were used as a template for in vitro DNA synthesis catalyzed by soluble protein extracts of E. coli. Viral strand DNA molecules isolated from fl RF DNA served as template as efficiently as did fl viral strand DNA isolated from phage particles (compare Fig. 1B and A). Approximately 60 pmol of product DNA was synthesized in 30 min in each case, representing near complete copying of the template DNA. Less than 2 pmol of product DNA was synthe-

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15 20 10 20 30 40 FRACTION NUMBER FIG. 2. Zone sedimentation analyses offl RFDNA product. fl viral strand DNA (450pmol) isolated from RF DNA was incubated in a standard initiation assay. After 30 min at 300C, the reaction was terminated by the addition of EDTA to 10 mM and Sarkosyl to 0.5%. The mixture was layered onto an isokinetic neutral pH sucrose gradient and spun for 4 h at 55,000 rpm at 150C in a Beckman SW56 rotor (A). Sedimentation is from right to left. The gradient was fractionated, and 10 Mul of the 0.2-ml fractions was analyzed for total radioactivity. The radioactivity in fractions 17 and above represents unincorporated [32P]TTP. The arrow marks the position of purified fl RFII DNA run in a parallel gradient. Pooled fractions (9, 10, 11) were dialyzed, and then 0.2 ml was layered onto an isokinetic alkaline pH sucrose 5

70 to

1^1 5

15

5 30 TIME (min)

15

30

FIG. 1. DNA synthesis directed by fl viral strand DNA. fl viral strand DNA (300 pmol), isolated from phage particles (A) or RF DNA (B), was incubated in a standard 50-Mul initiation assay. At the indicated times, 10-Mul aliquots were removed, and the amount of DNA synthesized was determined. DNA synthesis is expressed in picomoles ofproduct DNA per 10-,ul aliquot. Solid lines represent DNA synthesis in the absence of rifampin; dashed lines represent DNA synthesis in the presence of rifampin.

10

gradient. The tube was spun for 5 h at 55,000 rpm at 15°C in a Beckman SW56 rotor (B). The gradient was fractionated, and the total radioactivity was determined for each 0.1 -ml fraction. The arrow marks the position of unit-length linear fl viral strand DNA run in the same gradient.

DNA SYNTHESIS ON STRANDS OF fl RF DNA

VOL. 29,1979

site (M. Bayne and L. Dumas, unpublished data). Alkaline pH zone sedimentation analysis demonstrated that, whereas the product DNA strands made on both the viral strand template isolated from phage and from RF DNA were unit-length linear molecules prior to endonuclease treatment (Fig. 3A and C), cleavage by the enzyme resulted in product DNA molecules that sedimented as one-half unit length linears (Fig. 3B and D). This is the result expected if each fl RFII molecule contained a unique gap in the newly synthesized complementary strand located approximately 180°C away from the single endonuclease Bst 1503 cleavage site. This result is therefore consistent with a single chain initiation event occurring ort each template DNA molecule at this unique site. The initiation site for fl complementary strand DNA synthesis has been mapped on in vivo-replicating fl RF DNA (13). The location of this site is approximately 1800C away from the single endonuclease BamHI (or endonucle-

1017

ase Bst 1503) cleavage site on fl RF DNA (N. Zinder, personal communication). Based on these criteria, we conclude that the initiation of DNA synthesis on fl viral strand DNA isolated from intracellular RF DNA molecules is the same as the initiation of DNA synthesis on fl viral strand DNA isolated from phage particles. The initiation event is catalyzed by the same RNA polymerase and apparently occurs at the same site in vitro as used for the in vivo initiation of DNA synthesis. DNA synthesis on fl complementary strands isolated from intracellular RF DNA. Complementary strand circular DNA molecules isolated from fl RF DNA did not serve as template for DNA synthesis catalyzed by soluble protein extracts of E. coli (Fig. 4). Less than 2 pmol of product DNA was detected after 30 min both in the presence and absence of rifampin. DNA synthesis was observed on several other DNA templates under the same reaction conditions. In addition to catalyzing DNA synthesis of fl viral strand DNA, the soluble protein extract of E. coli was also able to catalyze DNA synthesis on phage G4 and 4X174 viral strand DNA molecules (Fig. 4). DNA synthesis on both G4 and sX174 DNA was rifampin resistant. DNA synthesis directed by 4sX174 DNA, but not G4 DNA, was dependent upon the activity of

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FIG. 3. Location of the gaps in fl RFII DNA. fl RFII molecules synthesized in a standard initiation assay, using either fl viral strand DNA isolated from phage or fl viral strand DNA isolated from RF, were isolated and digested with endonuclease Bst 1503 as described in the text. The DNA was then analyzed by isokinetic alkaline pH zone sedimentation. Arrows indicate the position of fl unit-length linear DNA absorbancy marker and the estmnated position for linear DNA of one-half unit length using Studier's approximation relating sedimentation coefficient to molecular weight (29). fl RFII synthesized using fl viral strand DNA from phage (A) before and (B) after endonuclease cleavage. fl RFII synthesized using fl viral strand DNA from RF (C) before and (D) after endonuclease cleavage.

Z

20 -,

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30 15 TIME (min) FIG. 4. DNA synthesis directed by fi complementary strand DNA. fl complementary strand DNA (270 pmol), dX174 viral strand DNA (300 pmol), or G4 viral strand DNA (300 pmol) was incubated in a standard 50-Itl initiation assay. At the indicated times, 10-ul aliquots were removed, and the amount of DNA synthesized was detennined. (-*) G4 DNA + rifampin; ()-- 0) 6X174 DNA + rifampin; (A-A) fl complementary strand DNA without rifampin; (A--- -A) fl complementary strand DNA + rifampin. 5

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BAYNE AND DUMAS

the E. coli dnaC protein. When soluble protein extracts prepared from E. coli PC79, a dnaC defective mutant, were used in the standard initiation assay, approximately 60 pmol of product DNA was synthesized in 30 min on G4 DNA, but less than 2 pmol of DNA was synthesized on 4X174 DNA in the presence or absence of rifampin.

Experiments similar to those described in Fig. 3 demonstrated that the DNA chain initiation event on G4 template DNA occurred at a single unique site (data not shown). This site is located at the same position as the single initiation site for G4 complementary strand DNA synthesis determined in vivo (19). In contrast, analysis of 4X174 RFII molecules synthesized in vitro demonstrated no unique site for the initiation of DNA synthesis on OX174 viral strand DNA (data not shown). This is consistent with other in vitro and in vivo analyses of the initiation of 4X174 complementary strand DNA synthesis which suggest that initiation can occur at any of several different sites (16, 18, 30). The addition of fl complementary strand DNA did not inhibit OX174 DNA, G4 DNA, or fl viral strand DNA-directed synthesis in soluble protein extracts of E. coli. This eliminated the possibility that an inhibitor present in the fl complementary strand preparation prevented DNA synthesis on this template. The above experiments indicate that the three known E. coli DNA chain initiation pathways are active and functioning with proper specificity under these reaction conditions: E. coli DNAdependent RNA polymerase-catalyzed primer synthesis on fl viral strand DNA; E. coli dnaG

protein-catalyzed primer synthesis on G4 viral strand DNA; and E. coli dnaB protein-dnaC protein-dependent primer synthesis on 4X174 DNA, catalyzed by the E. coli dnaG protein. Under these same conditions, fl complementary strand circular DNA molecules are not able to serve as templates for DNA synthesis. DNA synthesis on primed fl complementary strand DNA. We next asked if the failure of fl complementary strand circular DNA molecules to serve as templates for DNA synthesis in soluble protein extracts of E. coli was due to the inability to synthesize a primer oligonucleotide on this template, or to the inability of fl complementary strands to serve as templates for chain elongation reactions. We observed that fl complementary strand DNA was able to serve as template for DNA synthesis in a simple in vitro system using E. coli DNA polymerase I as chain elongation catalyst and small fragments of denatured calf thymus DNA as oligonucleotide primers (Fig. 5). Under the reaction conditions used, synthesis

limited to a onefold copy of the template DNA (6). These data demonstrated that fl complementary strand could be copied by a DNA polymerase when primed by small random oligonucleotides. This suggests that there is nothing inherent in the complementary strand that prevents its activity as a template in chain elongation reactions. We next tested the ability of the soluble protein extract of E. coli to copy fl complementary strand DNA which had been primed with a unique fragment of fl viral strand DNA. This primed template was prepared by annealing a specific 320-nucleotide fragment of fl viral strand DNA to the fl complementary DNA molecule as described in Materials and Methods. Figure 6A shows a neutral pH zone sedimenta-

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FIG. 5. Repair DNA synthesis at 15°C using E. coli DNA polymerase I. fl viral strand DNA (300 pmol) isolated from phage (A) and fl complementary strand DNA (270 pmol) isolated from RF (B) were incubated with E. coli DNA polymerase I and calf thymus DNA fragments in a final volume of 60 Ill as described in the text. At the indicated times, 10-I,I aliquots were removed, and the amount of DNA synthesis was determined. 25

(A)

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FRACTION NUMBER

FIG. 6. Zone sedimentation analyses of fl RF product synthesized using primed fl complementary strand DNA. Primed fl complementary strand DNA (380 pmol) was incubated in a standard initiation assay. The product DNA was analyzed as described in the legend to Fig. 2. (A) Zone sedimentation analysis at neutral pH; (B) zone sedimentation analysis ofpeak fractions (9, 10, 11) at alkaline pH.

DNA SYNTHESIS ON STRANDS OF fl RF DNA

VOL. 29, 1979

tion analysis of the product DNA synthesized. Essentially all of the product cosedimented with added fl RFII absorbancy marker. The material in the product DNA band (fractions 9, 10, and 11) was collected and subjected to further analyses. Figure 6B shows an alkaline pH zone sedimentation analysis of this material. These data demonstrated that full-length linear product DNA strands were synthesized on the primed fl complementary strand template. Further analyses of the DNA product in alkaline pH CsCl equilibrium gradients confirmed that the newly synthesized DNA strands were of fl viral strand density (Fig. 7B). These data demonstrate that the soluble protein extract of E. coli is able to copy the entire fl complementary strand template primed by a single, specific fragment of fl viral strand DNA. Therefore, there are no detectable elongation blocks on the complementary strand template. These data also demonstrate that our assay could detect DNA synthesis on fl complementary strand DNA if the de novo synthesis of an active primer oligonucleotide occurred on this template. Because of the high background of RNA synthesis unrelated to DNA replication in these soluble protein extracts of E. coli, it has not been possible to directly demonstrate primer oligonucleotide synthesis uncoupled from DNA chain elongation on active DNA templates (11). It was therefore not possible for us to directly demonstrate the inability of this system to synthesize a primer on fl complementary strand

1019

DNA. We can conclude from our data, however, that no detectable active primers were synthesized. Infectivity of ft complementary strand DNA. Since fl complementary strand DNA molecules did not serve as template for the detectable initiation of DNA synthesis in soluble protein extracts of E. coli, we wanted to determine if the E. coli cell could convert free circular fl complementary strands to duplex DNA. When incubated with E. coli spheroplasts, free circular viral strand DNA molecules can give rise to infectious phage particles (27). This might serve as an assay for in vivo initiation of DNA synthesis on single-stranded circular DNA molecules, since conversion to the duplex form is an essential step in phage production. Using this transfection assay, we compared the efficiencies of infection of fl viral and complementary strand circular DNA molecules (Table 1). In experiment I, circular viral strand fl DNA molecules had an efficiency of infection of 1.0 (3 x 10i PFU produced per ng of input DNA). Circular complementary strand fl DNA molecules had an efficiency of infection of 0.1. Strand analysis of the complementary strand preparation showed less than 5% contamination by viral strand DNA (1). These observations suggest, therefore, that E. coli spheroplasts are able to convert free fl complementary strand DNA to the duplex RF. However, the low efficiency of infection indicates that some step in the infection process (perhaps the conversion of input DNA to duplex RF DNA) is at best oneI

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FRACTION NUMBER FIG. 7. Equilibrium gradient analysis offl RFproduct. 3H-labeled fl RFII DNA synthesized using fl viral strand DNA isolated from phage, 3P-labeled fl RFII DNA synthesized using fl viral strand DNA isolated from RF, and 3P-labeledfl RFII DNA synthesized usingprimed fl complementary strand DNA were isolated as described in the legends to Fig. 2 and 6. The DNA molecules were then spun to equilibrium in an alkaline CsCI gradient as described in the text. Arrows mark the positions offl viral and complementary strand DNA absorbancy markers added to each gradient. (A) 3H-labeled fl RFII from viral strand (phage) (-4); 32p labeled fl RFII from viral strand (RF) (0 - -). (B) 3H-labeled fl RFII viral strand (phage) (@-4); 3plabeled fl RFII from primed complementary strand (0- - -0).

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J. VIROL.

BAYNE AND DUMAS TABLE 1. Infectivity offl DNAa

Expt

DNA type

PFU/ng of DNA

Efficiency tfionfc

I

Viral strand DNA (phage) Viral strand DNA (RF) strand Complementary DNA

3 x 104 3 x 104 3 x 103

1.0 1.0 0.1

tion

7 x 104 1.0 Viral strand DNA (phage) 0.1 strand 7 x 103 Complementary DNA 0.1 strand 7 x 103 Complementary DNA (primed) a The infectivity of fl viral strand DNA molecules isolated from phage particles, of fl viral and complementary strand DNA molecules isolated from RF DNA, and of fl complementary strand DNA molecules primed by a specific fragment of viral strand DNA was measured by using E. coli K-12 W6 spheroplasts as described in the text. Phage production was measured for dilutions of DNA from 1 ng to 100 pg. Efficiency of infection is defined as PFU per nanogram of input DNA divided by PFU per nanogram of fl viral strand DNA isolated from phage.

II

tenth as effective as when infecting with the free fl viral strand DNA. We next measured the infectivity of fl complementary strand DNA primed by a specific fragment of viral strand DNA. In soluble protein extracts of E. coli, these molecules were converted to RF DNA (Fig. 6). If chain initiation were in fact the limiting step in the infectivity assay, then primed fl complementary strand DNA should have a higher efficiency of infection than unprimed fl complementary strand DNA. However, in the transfection assay, primed fl complementary strand DNA also had an efficiency of infection of 0.1 (Table 1, experiment II). The presence of the potential primer fragment did not increase the infectivity of the fl complementary strand. Either chain initiation was not the limiting step in the infectivity assay, or the potential primer fragment could not be used. Given this observation, we are unable to say whether or not the reduced infectivity of the fl complementary strand DNA molecule was specifically due to its inability to serve as template for de novo primer synthesis in vivo. DISCUSSION The use of single-stranded circular DNA molecules as template for in vitro DNA synthesis provides a convenient assay for the de novo initiation of DNA synthesis. Soluble protein extracts of E. coli can detect DNA synthesis initiation sites recognized by E. coli DNA-dependent RNA polymerase (on bacteriophages fl, fd, and M13 DNA molecules), initiation sites recognized by the E. coli dnaG protein (on bacteriophage G4 DNA molecules), and DNA synthesis initiation sites requiring the activity of a multi-en-

zyme pre-initiation complex and the E. coli dnaG protein (on bacteriophage 4X174 DNA molecules). That this in vitro DNA synthesis faithfully reflects the initiation of DNA synthesis in vivo is evidenced by the dependence upon the same proteins and the use of the same initiation sites as determined for in vivo synthesis. We have used this assay system to examine the initiation of DNA synthesis on the isolated strands of bacteriophage fl RF DNA. When fl viral strand circular DNA molecules isolated from intracellular fl RF DNA were used as templates for DNA synthesis catalyzed soluble protein extracts of E. coli, DNA synthesis was rifampin sensitive, indicating the requirement for E. coli DNA-dependent RNA polymerase. The products of this synthesis were fl RFII molecules with unit-length linear complementary strands. The gap in the RFII molecules mapped at the same location as the DNA synthesis initiation site detected for in vivo-replicating RF molecules. We could detect no difference between DNA synthesis directed by fl viral strand DNA isolated from intracellular RF molecules and DNA synthesis directed by fl viral strand DNA isolated from phage particles. We conclude that both forms of fl viral strand DNA molecules carry the same DNA synthesis initiation site. When fl complementary strand circular DNA molecules isolated from intracellular fl RF DNA were used as templates for DNA synthesis catalyzed by soluble protein extracts of E. coli, no DNA synthesis was detected. The ability of the soluble protein extract to convert 4X174, G4, and fl viral strand DNA molecules to their duplex forms demonstrated the activities of the known E. coli DNA synthesis initiation proteins. The lack of DNA synthesis on fl complementary strand DNA was probably due to the absence of primer synthesis, since primed fl complementary strand DNA molecules were converted to duplex DNA under the same reaction conditions. Thus, unlike the 4X174, G4, and fl viral strand DNA molecules, fl complementary strand has no detectable site for de novo primer synthesis. Defective initiation of DNA synthesis on fl complementary strand DNA may also be responsible for the low infectivity of this DNA, although the transfection experiments failed to prove that this was the case. Studies of filamentous phage DNA synthesis in vivo suggest that RF replication occurs by the rolling circle mechanism (28, 31). The rolling circle model of DNA replication, in its simplest form, does not require the existence of de novo DNA synthesis initiation sites on the complementary strand template of the RF DNA mole-

VOL. 29, 1979

DNA SYNTHESIS ON STRANDS OF fl RF DNA

cule. Synthesis of new viral strand DNA can be primed by the 3'-OH end of the preexisting viral strand of the RF molecule produced by a specific endonuclease cleavage (12). The fl gene 2 protein is responsible for the introduction of a viral strand-specific nick in the fl RFI molecule (10, 15). It seems likely that in vivo fl viral strand DNA synthesis is initiated at this nick. Based on their examination of the origin and direction of fl DNA synthesis in vivo, Horiuchi and Zinder have proposed a simple model to explain fl RF replication (13). According to this model, the synthesis of viral and complementary strand DNA during fl RF replication is uncoupled; that is, RF replication is simply the sum of the asymmetric synthesis of free viral strand circular DNA molecules on the RF template by the rolling circle mechanism, and the subsequent conversion of these free viral strand circular DNA molecules to duplex RF DNA. The level of the fl gene 5 protein (a single-strand DNA binding protein) would regulate the switch between converting free viral strands to RF DNA molecules and packaging free viral strands into phage particles (20). This simple model necessitates no de novo initiation sites for DNA synthesis on the complementary strand of fl RF DNA. Viral strand DNA synthesis occurs by the rolling circle mechanism initiated by the fl gene 2 protein. In addition, this model suggests that a single de novo DNA synthesis initiation site of fl viral strand DNA, recognized by E. coli DNAdependent RNA polymerase, would be sufficient to account for all stages of fl DNA synthesis. A similar mode of replication may explain OX174 RF replication. Eisenberg et al. have recently reconstituted at least part of the 4X174 RF replication pathway (9). In this system, synthesis of new viral strand DNA occurs by the rolling circle mechanism by the extension of the nicked viral strand of the RFI template. The 4X174 cis A protein catalyzes this viral strandspecific cleavage. No de novo DNA synthesis initiation sites are detected on the complementary strand of the 4X174 RF molecule when using duplex DNA as template. Complementary strand DNA synthesis directed by the viral strand of the RF molecule is catalyzed by the same proteins used to convert free 4X174 viral strand circular molecules to duplex RF DNA. Our investigation of the initiation of DNA synthesis on the isolated strands of fl RF DNA provides independent evidence in support of the simple rolling circle model of RF replication. Under conditions where no preexisting viral strand DNA is present, we could detect no de novo DNA synthesis initiation sites on fl complementary strand DNA. In addition, our finding that fl viral strand DNA isolated from RF mol-

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ecules contains the same initiation site as fl viral strand DNA isolated from phage particles is in complete agreement with Horiuchi and Zinder's model for fl RF synthesis. Our studies do not explain the in vivo observations that filamentous phage of RF DNA replication is defective in E. coli dnaB and dnaG mutants. In fact, they raise the possibility that these host cell mutations have some indirect effect on filamentous phage RF DNA replication in vivo. The use of a more complex in vitro system, using intact fl RFI DNA and fl gene 2 protein, may be required to determine whether or not the protein products of these two genes are actually essential for the replication of the duplex RF DNA molecule. ACKNOWLEDGMENTS We are grateful to A. Kornberg, P. Carl, N. Zinder, and N. Godson for supplying bacterial strains and bacteriophages. We also thank J. Bieker and N. Welker for providing some of the enzymes used in this work. This research was supported by Public Health Service research grant AI-9882 and research career development award AI-70632 to L.B.D. from the National Institute of Allergy and Infectious Diseases. M.B. was supported in part by Public Health Service training grant GM-07291 from the National Institute of General Medical Sciences.

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